Eukaryotic cells undergo dynamic, carefully-orchestrated shape changes as they interact with their environment and respond to internal and external signals. For example, neutrophils squeeze through the walls of blood vessels and engulf unwelcome bacteria, and cell division is only complete after the physical separation of daughter cells. The networks of molecules that power and control these remarkably complex cell movements cannot be fully understood without detailed knowledge of the movements themselves. This proposal develops and demonstrates a Differential Force Microscope (DFM) that overcomes the single-cantilever limitations of Atomic Force Microscopy (AFM) to enable fundamentally new biophysical measurements of cell movements. AFMs were first developed to measure surface properties of inanimate samples - not to follow the complex movements of dynamic cells. Commercially-available AFMs use a single cantilever to measure one point at a time, preventing instantaneous comparison offered at different points and prohibiting real-time correction of measurements for instrument drift. This limitation on mechanical measurements of dynamic cell movements is overcome in the proposed instrument by operating two independent cantilevers simultaneously. The Aims of this work sequentially develop three specific measurement capabilities that will broadly benefit biophysical studies of cell movements: (1) measure absolute movements of a cell surface by normalizing instrument drift, (2) simultaneously measure forces exerted at two different points on a cell surface, and (3) stimulate one point on a cell and measure its mechanical response at a second point. For each aim, demonstration measurements are performed on fish keratocyte cells, a model system of cell motility. The resulting instrument will be made available to researchers interested in quantifying spatial and temporal coordination of cell movements.